The overall goal of this Program Project is to understand the molecular mechanisms involved in signal transduction during membrane trafficking and cell proliferation. Our interests are focussed on the role of receptor tyrosine kinases and on GTP-binding proteins of the ras and heterotrimeric G protein families and their upstream and downstream effectors. Project 1: The goal of this project is to determine the role of GTP-binding proteins (rabs, ARF, heterotrimeric G proteins) in the maintenance of Golgi organization and in the control of vesicular membrane traffic to and through the Golgi apparatus. These studies will make use of in vitro systems, permeabilized cells and microinjection. Project 2: This project involves the morphological characterization of the biochemical basis for the function of the rab and ARF families of ras related GTP binding proteins in vesicular transport between the ER and Golgi studied in intact and permeabilized cells. Project 3: This project takes advantage of the genetic approach and the availability of mutants defective in specific steps in vacuolar (lysosomal) targeting in yeast to identify proteins important in targeting. Two such mutants to be investigated have defective genes whose products appear to share sequence homology with Ser/Thr protein kinase and protein phosphatase 2A. Project 4: The purpose of this project is to define the sequence codes for occupancy induced internalization, sorting, down-regulation and recycling of the EGF receptor. The approach to be taken involves in vitro mutagenesis and production of stable transfectants expressing modified receptors. Project 5: Traffic along the endocytic, exocytic, and transcytotic pathways is mediated by vesicular carriers. This project proposes to isolate specific subpopulations of vesicular carriers and to identify and characterize their pilots and sorters. Project 6: The goal of this project is to define the molecular mechanisms which govern receptor-mediated endocytosis using a novel cell-free system. The biochemical requirements which distinguish ligand-induced internalization of the EGF receptor from the constitutive internalization of the transferrin receptor will be characterized and the molecular basis for these differences identified. Project 7: This project focuses on defining the glycosylation machinery in individual Golgi subcompartments of normal and transformed cells. The goals are to characterize the N- linked oligosaccharides synthesized by intact Golgi vesicles, to determine where oligosaccharides are assembled, and identify some of the requirements for assembly.